Project 2, Cell Cycle Checkpoints, will focus on checkpoint responses to UV-induced DMAdamage. Because cell cycle checkpoints enhance the repair of DNA damage, defects in checkpoint function enhance UV-induced chromosomal aberrations arid produce a"mutator" phenotype. UV induces and activates p53, which induces G1 arrest or apoptosis, it activates ATR to induce an intra-S checkpoint response to slow the ate of replicon initiation, and it triggers a p38 kinase-dependent G2 delay. Quantitative metrics will establish the functional capacities of normal human melanocytes for thesis checkpoint functions and whether melanoma lines display functional defects. Studies in Aim 1will express a dominant-negative p53 allele in normal human melanocytes to determine whether p53 signaling is required to arrest growth or induce apoptosis in response to UV damage. Studies in Aim 2 will determine whether melanocytes use the Rad17/ATR/Chk1 signaling pathway to inhibit replicon initiation in response to UV damage and whether knockdown of expression of a replication fork-protection complex composed of Timeless and Timeless- interacting protein inactivates the intra-S checkpoint response to UV. Studies in Aim 3 will determine whether expression of mutant B-Raf and N-Ras oncogenes in normal human melanocytes produces an attenuation of G2 checkpoint function. Melanocyte lines with selective inactivation of p16 and ARF will also be monitored to determine how inherited and somatic mutations in the CDKN2A/INK4A locus affect checkpoint responses to DNA damage. Microarray technology will be used to define signatures of basal gene expression that predict G1 and G2 checkpoint functions in melanoma lines and whether the signatures distinguish melanoma lines from lymph node and visceralmetastases. In Aim 4 a mathematical model of G2 checkpoint function will be developed to test how variation in levels of protein expression affect response outcomes. A computational model of UV-clastogenesis will be developed in Aim 4 to test how DNA repair and cell cycle checkpoint functions collaborate to protect against UV-induced chromosomal aberrations. Project 2 will enumerate cell cycle checkpoint function in normal human melanocytes, melanoma lines, and melanocytes with melanomagenic genetic alterations to determine whether defects in checkpoint function produce a chromosomal mutator phenotype to enhance UV-induced malignant progression.